A. The Blood-Brain Barrier
The blood-brain barrier (BBB) has been an impediment to successful drug delivery to the central nervous system (CNS). As a consequence, most diseases of the brain cannot be diagnosed and treated. Typically, only small-molecule drugs cross the BBB. That is why, for too long, the process of drug discovery has been centered on designing, developing and screening small molecules with activity at a particular site or receptor in the brain. However, small-molecule drugs for CNS targets have limitations, which include: i) non-specific targeting; ii) non-specific organ distribution; iii) low therapeutic indices; iv) development of drug resistance shortly after initial treatment; v) only a small percentage of small-molecule drugs cross the BBB and vi) only 1% of the total number of drugs were active in the CNS [Pardridge W M, NeuroRX, 2 (1), 3 (2006)]. In addition, only a few diseases of the brain, such as depression, chronic pain, and epilepsy, respond to this category of small molecules. Most serious diseases of the brain such as Alzheimer's disease (AD); Parkinson's disease (PD); brain cancer; stroke; brain and spinal cord injury; HIV infection of the brain; Huntington disease; multiple sclerosis (MS); and many childhood inborn genetic errors affecting the brain do not respond to small molecule drugs, irrespective of the lipid solubility of the drug. A handful of FDA approved small molecule drugs, e.g. Aricept (AD), Cognex (AD), Exelon (AD), Razadyne (AD), and Levodopa (PD), for neurodegenerative diseases that slow down the disease symptoms in some patients stop working after a period of time, leaving the patient to helplessly succumb to his/her disease.
Development of large molecule drugs is generally discouraged because of their typically poor BBB permeability. Many potential large molecule modern drugs, such as, engineered proteins (e.g.: nerve growth factors), antibodies, genes, vectors, micro-RNA, siRNA, oligonucleotides and ribozymes, which are otherwise effective in ex-vivo studies, have not been developed for clinical use due to a failure to deliver them in sufficient quantity into the CNS. Although Alzheimer's disease (AD) has been known for more than a century and despite enormous research efforts both by private sectors and government institutes, there are no diagnostics or curative treatments for diseases of the CNS. More than 55 million people (and 6.5 million Americans in the US) are afflicted worldwide by neurodegenerative diseases (Alzheimer's disease and Parkinson's disease are the most common forms of degenerative dementia). These troubling statistics demonstrate an unmet need of developing technologies to solve the issues of diagnosing and treating neurodegenerative and tumor diseases in the CNS.
The BBB is formed by tight junctions between the cerebral endothelial cells, which are produced by the interaction of several transmembrane proteins that project into and seal the paracellular pathways (FIG. 1). The interaction of these junctional proteins, particularly, occludin and claudin, is complex and effectively blocks an aqueous route of free diffusion for polar solutes from blood along these potential paracellular pathways and thus denies these solutes free access to cerebrospinal fluid. Major scientific efforts over the years have led to the development of the following methods to cross the BBB: (i) The use of liposomes or other charged lipid formulations, which have limited complex stability in serum and high toxicity over time (Whittlessey K J et al., Biomaterials, 27, 2477 (2006)); (ii) Electroporation-based techniques which are only effective when performed during a specific window of development in healthy cells, with eventual loss of expression or bioactivity (Gartner et al., Methods Enzymology 406, 374 (2006)), and (iii) Viral-based vectors and fusions which have shown only limited efficacy in humans and animals while raising a number of safety concerns, and typically requiring invasive procedures such as direct injection into the brain to achieve targeted delivery (Luo D, Nat Biotechol, 18 (8), 893 (2000)). Thus, there is an unmet need to develop novel technologies to breach the BBB.
B. Strategies for Drug Delivery Across the Blood-Brain Barrier
Invasive strategies such as intra-cerebroventricular infusion, convection-enhanced delivery, and intra-cerebral Injection are covered in the following references: Pardridge W M, Pharma Res., 24, 1733 (2007); Pardridge W M, Neuro RX, 2, 3 (2005); Vandergrift W A, et al., Neurosurg Focus, 20, E10 (2006); Funk L K, et al., Cancer Res., 58, 672 (1998); Marks W J, et al., Lancet Neurol, 7, 400 (2008); and Herzog C D, et al., Mov. Disord, 22, 1124 (2007).
Disruption of the BBB using bradykynin analogues, ultrasound, and osmotic pressure are covered in the following references: Borlogan C V, et al., Brain Research Bulletin, 60, 2970306 (2003); Hynynen K, et al., J. Neurosurg., 105, 445 (2006); and Fortin D, et al., Cancer, 109, 751 (2007).
Physiological approaches involving transporter-mediated delivery, receptor-mediated transcytosis, adsorptive-mediated transcytosis are covered in the following references: Allen D D, et al., J. Pharmacol Exp Ther, 304, 1268 (2003); Coloma M J, et al., Pharm Res, 17, 266 (2000); Jones A R, et al., Pharma Res, 24, 1759 (2007); Boada R J, et al., Biotech Bioeng, 100, 387 (2007); Pardridge W M, Pharma Res, 3, 90 (2003); Zhang Y, et al., J. Pharmaco Exp Therap, 313, 1075 (2005); and Zhang Y, et al, Brain Res, 1111, 227 (2006).
Pharmacological approaches involving chemical modification of drugs to lipophilic molecules or encapsulation into liposomes are covered by the following references: Bradley M O, Webb N L, et al., Clin. Cancer Res., 7, 3229 (2001); Lipinski C A, Lombardo F, et al., Adv. Drug Deliv Rev, 46, 3 (2001); Huwyler J, et al., J. Pharmacol Exp Ther, 282, 1541 (1997); Madrid Y, et al., Adv Pharmacol, 22, 299 (1991); Huwyler J, Wu D, et al., Proc. Natl. Acad. Sci. USA, 93, 14164 (1996); Swada G A, et al., J. Pharmacol Exp Ther, 288, 1327 (1999); and Shashoua V E, et al., Life Sci., 58, 1347 (1996).
Resistance to opsonization and nanoparticles based drug delivery across the BBB, whereby the drug is passively adsorbed on to the particles, is covered by following references: Greiling W, Ehrlich P, Verlag E, Dusseldorf, Germany, p. 48, 1954; Couvreur P, Kante B, et al., J. Pharm Pharmacol, 31, 331 (1979); Douglas S J, et al., J. Colloid. Interface Sci, 101, 149 (1984); Douglas S J, et al., J. Colloid Interface Sci., 103, 154 (1985); Khanna S C, Speiser P, J. Pharm. Sci, 58, 1114 (1969); Khanna S C, et al., J. Pharm. Sci, 59, 614 (1970); Sugibayashi K, et al., J. Pharm. Dyn, 2, 350 (1979b); Brasseur F, Couvreur P, et al., Actinomycin D absorbed on polymethylcyanoacrylate: increased efficiency against an experimental tumor, Eur. J. Cancer, 16, 1441 (1980); Widder K J, et al., Eur. J. Cancer, 19, 141 (1983); Couvreur P, et al., Toxicity of polyalkylcyanoacrylate nanoparticles II. Doxorubicin-loaded nanoparticles, J. Pharma Sci, 71, 790 (1982); Couvreur P, et al., Biodegradable polymeric nanoparticles as drug carrier for antitumor agents, Polymeric Nanoparticles and Microspheres, CRC Press, Boca Raton, pp. 27-93 (1986); Grislain L, Couvreur P, et al., Pharmacokinetics and distribution of a biodegradable drug-carrier, Int. J. Pharm., 15, 335 (1983); Mukherjee P, et al., Potential therapeutic applications of gold nanoparticles in BCLL, J. Nanobiotechnology, 5, 4 (2007); Maeda H and Matsumura Y, Tumoritropic and lymphotropic principles of macromolecular drugs, Crit. Rev. Ther. Drug Carrier Syst., 6, 193 (1989); Kattan, J et al., Phase I clinical trial and pharmacokinetic evaluation of doxorubicin carried by polyisohexylcyanoacrylate nanoparticles, Invest. New Drugs, 10, 191 (1992); Kreuter J, Naoparticles—A historical perspective, Int. J. Pharm., 331, 1 (2007); Alyautdin R, et al., Analgesic activity of the hexapeptide dalargin adsorbed on the surface of polysorbate 80-coated poly(butyl cyanoacrylate) nanoparticles. Eur. J. Pharm. Biopharm., 41, 44 (1995); Kreuter J, Alyautdin R, et al., Passage of peptides through the blood-brain barrier with colloidal polymer particles (nanoparticles), Brain Res., 674, 171 (1995); Alyautdin R N et al., Delivery of loperamide across the blood-brain barrier with polysorbate 80-coated polybutylcyanoacrylate nanoparticles, Pharm. Res., 14, 325 (1997); Schroeder U, et al., Body distribution of 3H-labeled dalargin bound to polybutylcyanoacrylate, Life Sci., 66, 495 (2000); Alyautdin R N et al., Significant entry of tubocurarine into the brain of rats by absorption to polysorbate 80-coated polybutyl-cyanoacrylate nanoparticles: an in situ brain perfusion study, J. Microencapsul., 15, 67 (1998); Gulyaev A E, Gelperina S E, et al., Significant transport of doxorubicin into the brain with polysorbate 80-coated nanoparticles, Pharm. Res., 16, 1564 (1999); Steiniger S C J, Kreuter J, et al., Chemotherapy of glioblastoma in rats using doxorubicin-loaded nanoparticles, Int. J. Cancer, 109, 759 (2004); Hekmatara T, et al., Efficient systemic therapy of rat glioblastoma by nanoparticle-bound doxorubicin is due to antiangiogenic effects, Clin. Neuropath., 28, 153 (2009); Gelperina S E, et al., Toxicological studies of doxorubicin bound to polysorbate 80-coated polybutylcyanoacrylate nanoparticles in healthy rats and rats with intracranial glioblastoma, Toxicol. Lett., 126, 131 (2002); Couvreur P, et al., J. Pharm. Sci, 71, 790 (1982); Kreuter J, et al., Apolipoprotein-mediated transport of nanoparticles-bound drugs across the blood-brain barrier, J. Drug Targeting, 10, 317 (2002); Davis S S, Biomedical appplications of nanotechnology-implications for drug targeting and gene therapy, Tibtech, 15, 217 (1997); Moghimi S M, Szebeni J, Stealth liposome and long circulating nanoparticles: Critical issues in pharmacokinetics, opsonization and protein-binding properties, Progress in Lipid Research, 42, 463 (2003); Arvizo R R, et al., Modulating pharmacokinetics, tumor uptake and biodistribution by engineered nanoparticles, PLos One, 6, e24374 (2011); Kurakhmaeva K B, et al., Brain targeting of nerve growth factor using polybutylcyanoacrylate nanoparticles, J. Drug Targeting, 17, 564 (2009); and Reukov V, et al., Proteins conjugated to polybutylcyanoacrylate nanoparticles as potential neuroprotective agents, Biotechnology and Bioengineering, 108, 243 (2010).
Shortcomings of Nanoparticles.
Although nanoparticles have made significant contributions to the field of medical sciences, most of the published studies have been conducted with drugs non-covalently coated to nanoparticles, thereby perhaps not realizing the full potential of nanomedicine.
C. Single-Domain Antibodies
In 1983 it was reported that the sera of camelid contained two different kinds of immunoglobulin: conventional heterodimeric IgGs composed of heavy and light chains, and unconventional IgGs without the light chains [Grover Y P, et al., Indian Journal of Biochemistry and Biophysics, 20, 238 (1983)]. Grover et al. demonstrated the presence of three bands which were designated as IgM, IgA, and a broad heterogeneous band containing a mixture of IgG complexes. One can speculate that the broad band these authors observed was due to the presence of mixture of normal IgG with a molecular weight (MW) of ˜160 KDa and heavy-chain IgG, without the light chain, with a MW of ˜80 KDa. However, since these authors did not use a proper sizing marker, the broad IgGs band could not be satisfactorily characterized.
Ungar-Waron et al. disclosed a SDS-PAGE analysis of camelid IgGs mixture treated with and without 2-mercaptoethanol (2ME) [Israel J. Vet. Medicine, 43 (3), 198 (1987)]. In the absence of 2-ME, IgG-complex, obtained from camelid serum, dissociated into two components with approximate molecular weight (MW) of 160 KDa (Conventional IgG) and ˜100 KDa (New IgG) on SDS-PAGE. However, in the presence of 2-ME, three bands of MW 55 KDa (gamma-like heavy-chain), 22 KDa (Light chain) and an additional protein band of 42 KDa (now known as heavy-chain only camelid antibody band without the light chains) were seen.
Subsequently, Azwai et al. from University of Liverpool, UK, independently confirmed the presence of an additional IgG band in camelid serums with a molecular weight of 42 KDa by SDS-PAGE electrophoresis under reducing conditions [Azwai, S. M., et al., J. Comp. Path., 109, 187 (1993)].
Hamers-Casterman et al. also reported similar findings, confirming independently the presence of 42 KDa IgG subclass in the sera of camelids upon SDS-PAGE analysis under reducing conditions [Hamers-Casterman et al., Nature, 363, 446 (1993) and U.S. Pat. No. 6,005,079].
Thus, two types of antibodies exist in camels, dromedaries, and llamas: one a conventional hetero-tetramer having two heavy and two light chains (MW ˜160 KDa), and the other consisting of only two heavy chains, devoid of light chains (MW ˜80 to 90 KDa).
In addition to camelid antibodies having only two heavy chains and devoid of light chains, a distinctly unconventional antibody isotype was identified in the serum of nurse sharks (Ginglymostoma cirratum) and wobbegong sharks (Orectolobus maculatus). The antibody was called the Ig new antigen receptors (IgNARs). They are disulfide-bonded homodimers consisting of five constant domains (CNAR) and one variable domain (VNAR). There is no light chain, and the individual variable domains are independent in solution and do not appear to associate across a hydrophobic interface [Greenberg A S, Avila D, Hughes M, Hughes A, McKinney E, Flajnik M F, Nature 374, 168 (1995); Nuttall S D, Krishnan U V, Hattarki M, De Gori R, Irving R A, Hudson P J, Mol. Immunol., 38, 313 (2001), Comp. Biochem. Physiol. B., 15, 225 (1973)]. There are three different types of IgNARs characterized by their time of appearance in shark development, and by their disulfide bond pattern [Diaz M, Stanfield R L, Greenberg A S, Flajnik, M F, Immunogenetics, 54, 501 (2002); Nuttall S D, Krishnan U V, Doughty L, Pearson K, Ryan M T, Hoogenraad N J, Hattarki M, Carmichael J A, Irving R A, Hudson P J, Eur. J. Biochem. 270, 3543 (2003)].
The natural hetero-tetrameric structure of antibodies exists in humans and most animals. The heavy-chain only dimer structure is considered natural characteristic of camelids and sharks [Holliger P, Hudson P J, Nature Biotechnology, 23, 1126 (2005)]. These antibodies are relatively simple molecules but with unique characteristics. Since the variable antigen binding (Vab) site binds its antigen only through the heavy-chain, these antibodies are also known as single-domain antibodies (sd-Abs). Their size is about one-half the size of traditional tetrameric antibodies, hence a lower molecular weight (˜80 KDa to 90 KDa), with similar antigen binding affinity, but with water solubility 100- to 1000-fold higher than conventional antibodies.
Another characteristic of heavy-chain antibodies derived from sharks and camelids is that they have very high thermal stability compared to the conventional mAbs. For example, camelid antibodies can maintain their antigen binding ability even at 90° C. [Biochim. Biophys. Acta., 141, 7 (1999)]. Furthermore, complementary determining region 3 (CDR3) of camelid Vab region is longer, comprising 16-21 amino acids, than the CDR3 of mouse VH region, comprising 9 amino acids [Protein Engineering, 7, 1129 (1994)]. The larger length of CDR3 of camelid Vab region is responsible for higher diversity of antibody repertoire of camelid antibodies compared to conventional antibodies.
In addition to being devoid of light chains, the camelid heavy-chain antibodies also lack the first domain of the constant region called CH1, though the shark antibodies do have a CH1 domain and two additional constant domains, CH4 and CH5 [Nature Biotech. 23, 1126 (2005)]. Furthermore, the hinge regions (HRs) of camelid and shark antibodies have an amino acid sequence different from that of normal heterotetrameric conventional antibodies [Muyldermans S, Reviews in Mol. Biotech., 74, 277 (2001)]. Without the light chain, these heavy-chain antibodies bind to their antigens by one single domain, the variable antigen-binding domain of the heavy-chain immunoglobulin, which is referred to as Vab in this application (VHH in the literature), to distinguish it from the variable domain VH of the conventional antibodies.
The single-domain Vab is surprisingly stable by itself, without having to be attached to the heavy-chain. This smallest intact and independently functional antigen-binding fragment Vab, with a molecular weight of ˜12-15 KDa, derived from a functional heavy-chain only full length IgG, is referred to as a “nanobody” In the literature [Muyldermans S, Reviews in Mol. Biotech., 74, 277 (2001)].
The genes encoding these full length single-domain heavy-chain antibodies and the antibody-antigen binding fragment Vab (camelid and shark) can be cloned in phage display vectors, and selection of antigen binders by panning and expression of selected Vab in bacteria offer a very good alternative procedure to produce these antibodies on a large scale. Also, only one domain has to be cloned and expressed to produce in vivo an intact, matured antigen-binding fragment.
There are structural differences between the variable regions of single domain antibodies and conventional antibodies. Conventional antibodies have three constant domains while camelid has two and shark has five constant domains. The largest structural difference is, however, found between a VH (conventional antibodies) and Vab (heavy-chain only antibodies of camelid and shark) (see below) at the hypervariable regions. Camelid Vab and shark V-NAR domains each display surface loops which are larger than for conventional murine and human IgGs, and are able to penetrate cavities in target antigens, such as enzyme active sites and canyons in viral and infectious disease biomarkers [Proc. Natl. Acad. Sci. USA., 101, 12444 (2004); Proteins, 55, 187 (2005)]. In human and mouse the VH loops are folded in a limited number of canonical structures. In contrast, the antigen binding loop of Vab possess many deviations of these canonical structures that specifically bind into such active sites, therefore, represent powerful tool to modulate biological activities [K. Decanniere et al., Structure, 7, 361 (2000)]. The high incidence of amino acid insertions or deletions, in or adjacent to first and second antigen-binding loops of Vab will undoubtedly diversify, even further, the possible antigen-binding loop conformations.
Though there are structural differences between camelid and shark parent heavy-chain antibodies, the antigen-antibody binding domains, Vab and V-NAR, respectively, are similar. The chemical and/or protease digestion of camelid and shark antibodies results in Vab and V-NAR domains, with similar binding affinities to the target antigens [Nature Biotech., 23, 1126 (2005)].
Other structural differences are due to the hydrophilic amino acid residues which are scattered throughout the primary structure of Vab domain. These amino acid substitutions are, for example, L45R, L45C, V37Y, G44E, and W47G. Therefore, the solubility of Vab is much higher than the Fab fragment of conventional mouse and human antibodies.
Another characteristic feature of the structure of camelid Vab and shark V-NAR is that it often contains a cysteine residue in the CDR3 in addition to cysteines that normally exist at positions 22 and 92 of the variable region. The cysteine residues in CDR3 form S—S bonds with other cysteines in the vicinity of CDR1 or CDR2 [Protein Engineering, 7, 1129 (1994)]. CDR1 and CDR2 are determined by the germline V gene. They play important roles together with CDR3 in antigenic binding [Nature Structural Biol., 9, 803 (1996); J. Mol. Biol., 311, 123 (2001)]. Like camelid CDR3, shark also has elongated CDR3 regions comprising of 16-27 amino acids residues [Eur. J. Immunol., 35, 936 (2005)].
The germlines of dromedaries and llamas are classified according to the length of CDR2 and cysteine positions in the V region [Nguyen et al., EMBO J., 19, 921 (2000); Harmsen et al., Mol. Immun., 37, 579 (2000)].
Immunization of camelids with enzymes generates heavy-chain antibodies (HCAb) significant proportions of which are known to act as competitive enzyme inhibitors that interact with the cavity of the active site [M. Lauwereys et al., EMBO, J. 17, 3512 (1998)]. In contrast, the conventional antibodies that are competitive enzyme inhibitors cannot bind into large cavities on the antigen surface. Camelid antibodies, therefore, recognize unique epitopes that are out of reach for conventional antibodies.
Production of inhibitory recombinant Vab that bind specifically into cavities on the surface of variety of enzymes, namely, lysozyme, carbonic anhydrase, alfa-amylase, and beta-lactamase has been achieved [M. Lauwereys, et al., EMBO, J. 17, 3512 (1998)]. Hepatitis C protease inhibitor from the camelised human VH has been isolated against an 11 amino. Eng. acid sequence of the viral protease [F. Martin et al., Prot, 10, 607 (1997)].